- Much research today centers around DNA (deoxyribonucleic acid) and how it determines physical traits and regulates body functions. Even though we have had nearly 50 years of research on DNA, much remains to be learned, and every answer seems to create more questions.
- In this lab, we will be using the chemical properties of DNA, and its attraction to something polar, to extract it from the cells of onions.
MATERIALS:
crushed Ice
Knife
Onion
Isopropyl alcohol (rubbing alcohol)
tap water
120mL Distilled water
1.5g Sodium chloride NaCl (table salt)
5.0g Sodium hydrogen carbonate NaHCO3 (baking soda)
5.0mL Liquid laundry detergent
transfer pipette (eye dropper)
250-mL erlenmeyer flask
Stirring rod
Filter paper
Filter funnel
Ring stand with small ring to hold funnel
Two 1000-mL beakers (for ice baths)
100-mL beaker
2 test tubes
Test tube rack
Mortar and pestlePROCEDURE:
- Steps 1 through 3 should be done ahead of time if possible so the solutions will be nearly zero degrees C. when they are used.
1. Prepare a buffer solution by mixing the following in a 250-mL erlenmeyer flask.
- 120mL Distilled water
- 1.5g Sodium chloride NaCl (table salt)
- 5.0g Sodium hydrogen carbonate NaHCO3 (baking soda)
- 5.0mL Liquid laundry detergent
- The buffer solution has several purposes in this lab. The saltiness and acidity (pH) of the solution is very close to that of living cells, so the DNA will redily dissolve into this solution. The detergent is added to help break down the cell walls of the onion cells. Cell walls are made of long polar molecules with a "greasy" end and a charged end. The detergent attaches to the "greasy" ends of these molecules, and pulls them apart. The cell walls must be broken down, so we can get to the DNA inside the cells.
2. Chill the buffer solution by placing the flask in a 1000-mL beaker half filled with crushed ice and tap water.
- We chill the buffer solution, because DNA molecules are fragile, and break apart easily when removed from the cell. By chilling the buffer solution to near freezing, we slow the rate of break up of the DNA.
3. Prepare a second ice water bath, and place the bottle of Isopropyl alcohol into it.
4. Dice an onion with a knife. Half an onion should be plenty for this lab. Use a mortar and pestle to mash the pieces of onion into a pulp. This brakes as many cell walls as possible.
5. Place 10.0mL of the onion pulp/juice into a 100-mL beaker and add 20mL of the chilled buffer solution. Stir vigorously with a stirring rod for three minutes. The detergent will break up the cell walls, releasing the onion DNA into the buffer solution.
6. Pour as much liquid as you can into a clean test tube. Let the test tube sit in your ice/water bath for five minutes. The solids should settle to the bottom of the test tube, and the top should mainly be liquid.
7. Set up the funnel in the ring stand with the filter paper. Place the test tube rack with another test tube under the funnel to catch the solution. Decant the liquid through the filter paper, retaining the pulp. The test tube should be about half full. If you have more than this, pour some out.
- The clear solution you have in this test tube consists of dissolved DNA fragments, as well as some other biochemical compounds such as RNA and proteins. DNA is a very long molecule, but compared to the holes in a piece of filtering paper, the molecule is still small enough to pass through.
8. Obtain some ice-cold isopropyl alcohol from the ice bath or a freezer. Using the transfer pipette, gently add alcohol to the top until there is about an inch and a half sitting above the buffer solution. The best way to do this is to let the alcohol run slowly down the side of the test tube and onto the DNA solution. You want as little mixing of the two liquids as possible.
- The rubbing alcohol is used to extract the DNA from the onion juice. The reason you want the rubbing alcohol to stay on top of the onion juice is because by doing that the liquid will form two distinct layers. Generally, molecules are attracted to the boundaries of two liquids. Sometimes the concentration of large molecules can be much higher at the boundary between two liquids. That's what we're hoping for here... if the DNA is attracted to the surface, we can pull most of it out. However, if the alcohol and onion juice mixes too much, there will be too much alcohol throughout the whole liquid, and the DNA won't be attracted to the surface, making it much harder to pull any out of the tube.
9. Very gently insert a glass rod through the upper alcohol layer in the test tube into the DNA containing buffer solution. While disturbing the solution as little as possible, leave the glass rod in one place and rotate it in one direction. With luck the DNA fragments will wind onto the glass rod in the same way that thread winds onto a spool.
- DNA spools onto the glass rod because the exposed ends have polar chemical groups on them. Glass is also polar, so the ends of the DNA are attracted to the rod. By winding the rod, you are basically just reeling in the DNA molecules.
10. After twirling the rod for about 60 seconds, pull the rod up through the alcohol layer. You should see the DNA adhere to the end of the rod and appear as a transparent, viscous sludge at its tip. The molecule that you have collected on this stick consists of the entire genetic code for the making of an onion.
- When you pull the DNA through the nonpolar alcohol layer, it clumps together because it is attracted to polar materials such as the rod or itself and not the alcohol.
11. Clean up.
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Back to Lab Dad's Laboratory
- In this lab, we will be using the chemical properties of DNA, and its attraction to something polar, to extract it from the cells of onions.
EXTRACTING DNA
FROM AN ONION
INTRODUCTION: